The class I A phosphoinositide 3-kinases (PI3Ks) consist of a 110-kDa catalytic domain and a regulatory subunit encoded by the p85␣, p85␤, or p55␥ genes. We have determined the effects of disrupting the p85␣ gene on the responses of mast cells stimulated by the cross-linking of Kit and Fc⑀RI, receptors that reflect innate and adaptive responses, respectively. The absence of p85␣ gene products partially inhibited Kit ligand/stem cell factor-induced secretory granule exocytosis, proliferation, and phosphorylation of the serine/threonine kinase Akt. In contrast, p85␣ gene products were not required for Fc⑀RI-initiated exocytosis and phosphorylation of Akt. LY294002, which inhibits all classes of PI3Ks, strongly suppressed Kit-and Fc⑀RI-induced responses in p85␣ ؊/؊ mast cells, revealing the contribution of another PI3K family member(s). In contrast to B lymphocytes, mast cell proliferation was not dependent on Bruton's tyrosine kinase, a downstream effector of PI3K, revealing a distinct pathway of PI3K-dependent proliferation in mast cells. Our findings represent the first example of receptor-specific usage of different PI3K family members in a single cell type. In addition, because Kit-but not Fc⑀RI-initiated signaling is associated with mast cell proliferation, the results provide evidence that distinct biologic functions signaled by these two receptors may reflect differential usage of PI3Ks.Mast cells (MCs) 1 are functionally dynamic effector cells of innate and adaptive immunity (1). Two MC surface receptors, namely, the Kit receptor (the product of the c-kit proto-oncogene) and the high affinity receptor for IgE (Fc⑀RI), provide activation via innate and adaptive immune mechanisms, respectively (2-4). Kit is a receptor tyrosine kinase belonging to the colony-stimulating factor-1/platelet-derived growth factor receptor subfamily (3). Kit is encoded by the murine White Spotting (W) locus (5, 6) and controls various cellular events during development and in adult life. Mutations at the W locus result in defects in gametogenesis, melanogenesis, and hematopoiesis (7,8). The hematopoietic defects include macrocytic anemia (8) and the virtual absence of tissue mast cells (9). Kit is expressed on both mature MCs and their earliest progenitors (10) as well as on cells of erythroid and melanocytic lineages and on germ cells (11). Kit ligand (KL; also known as stem cell factor), is expressed in membrane-associated and soluble forms (12) by mast cells (13), fibroblasts (11), endothelial cells (14), stromal cells (15), keratinocytes (16), neuroblastoma cells (17), and tumor cell lines (18). Although KL represents a major growth and differentiation factor for both murine and human MCs (19,20), it also promotes Kit-dependent MC mediator release (21-23), as well as enhances the release of MC mediators via IgE-dependent mechanisms (22,24). Fc⑀RI belongs to the antigen receptor superfamily (4). Rodent Fc⑀RI is a tetrameric receptor consisting of an ␣ chain, ␤ chain, and a dimer of disulfide-linked ␥ chains, whereas human Fc⑀...

We define by molecular, pharmacologic, and physiologic approaches the proximal mechanism by which the immunoglobulin superfamily member gp49B1 inhibits mast cell activation mediated by the high affinity Fc receptor for IgE (Fc⑀RI). In rat basophilic leukemia-2H3 cells expressing transfected mouse gp49B1, mutation of tyrosine to phenylalanine in either of the two immunoreceptor tyrosine-based inhibitory motifs of the gp49B1 cytoplasmic domain partially suppressed gp49B1-mediated inhibition of exocytosis, whereas mutation of both abolished inhibitory capacity. Sodium pervanadate elicited tyrosine phosphorylation of native gp49B1 and association of the tyrosine phosphatases src homology 2 domain-containing phosphatase-1 (SHP-1) and SHP-2 in mouse bone marrow-derived mast cells (mBMMCs). SHP-1 associated transiently with gp49B1 within 1 min after coligation of gp49B1 with cross-linked Fc⑀RI in mBMMCs. SHP-1-deficient mBMMCs exhibited a partial loss of gp49B1-mediated inhibition of Fc⑀RI-induced exocytosis at concentrations of IgE providing optimal exocytosis, revealing a central, but not exclusive, SHP-1 requirement in the counter-regulatory pathway. Coligation of gp49B1 with cross-linked Fc⑀RI on mBMMCs inhibited early release of calcium from intracellular stores and subsequent influx of extracellular calcium, consistent with SHP-1 participation. Because exocytosis is complete within 2 min in mBMMCs, our studies establish a role for SHP-1 in the initial counter-regulatory cellular responses whereby gp49B1 immunoreceptor tyrosine-based inhibition motifs rapidly transmit inhibition of Fc⑀RI-mediated exocytosis.The cross-linking of the high affinity Fc receptor for IgE (Fc⑀RI) 1 on the surface of mast cells activates intracellular signaling pathways that lead to exocytosis of preformed secretory granule components, generation of membrane-derived lipid mediators, and elaboration of immunoregulatory and proinflammatory cytokines. Through the release of their array of bioactive mediators, mast cells have an unusually high proinflammatory potential and contribute to host defense in animal models (1-3) and to the pathogenesis of allergic reactions and bronchial asthma in humans. In addition to Fc⑀RI, mouse mast cells express on their surface gp49B1 (4, 5), an immunoglobulin (Ig) superfamily member with two Ig-like domains (6) and two cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs), which have the amino acid sequence (I/V/S)XYXX(L/V) (7-12). We have shown that when rat IgE bound to Fc⑀RI on mouse interleukin-3-dependent, bone marrow-derived mast cells (mBMMCs) is both cross-linked to itself and coligated with rat anti-gp49B1 mAb bound to gp49B1, by means of F(abЈ) 2 mouse anti-rat IgG (heavy and light chain reactive), the release of secretory granule-and lipid-derived mediators is inhibited (9). As defined by ALIGN amino acid homology analysis, gp49B1 belongs to a family of receptors that has immunoregulatory potential (9, 13). In the mouse, the sole other member of this homology-based family (14) that...